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Improving the Thermostability of Glutamate Decarboxylase from Lactobacillus brevis by Consensus Mutagenesis

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Abstract

γ-Aminobutyrate (GABA) is an important bioactive compound synthesized through decarboxylation of L-glutamate by the glutamate decarboxylase (GAD). In this study, stabilized variants of the GAD from Lactobacillus brevis were constructed by consensus mutagenesis. Using Consensus Finder (http://cbs-kazlab.oit.umn.edu/), eight positions with the most prevalent amino acid (over 60% threshold) among the homologous family members were identified. Subsequently, these eight residues were individually mutated to match the consensus sequence using site-directed mutagenesis. Compared to the wild-type, T383K variant displayed the largest shift in thermostability among the single variants, with a 3.0 °C increase in semi-inactivation temperature (T5015), a 1.7-fold improvement of half-life (t1/2) at 55 °C, and a 1.2-fold improvement of t1/2 at 37 °C, respectively, while its catalytic efficiency (kcat/Km) was reduced. To obtain the mutant with improvement in both thermostability and catalytic activity, we performed a site-saturation mutation at T383. Notably, mutants T383V and T383G exhibited an increasement in thermostability and kcat/Km than that of wild-type. This study not only emphasizes the value of consensus mutagenesis for improving the thermostability of GAD but also sheds a powerful guidance to study the thermal stability of other enzymes.

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Funding

This study was funded by the National Natural Science Foundation of China (Nos. 31470793, 31670804, and 31971372) and Zhejiang Natural Science Foundation (Nos. LZ13B060002, LY16B060008, and LQ18B060002); and Innovation Fund for Graduate students of Zhejiang University of Science and Technology (2019YJSKC08).

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Author notes

  1. Yujiao Hua and Changjiang Lv contributed equally to this work.

Authors and Affiliations

  1. School of Biological and Chemical Engineering, Zhejiang University of Science and Technology, Hangzhou, 310023, China

    Yujiao Hua, Changjiang Lyu, Chunyan Liu, Hongpeng Wang & Jun Huang

  2. School of Biotechnology and Chemical Engineering, Ningbo Institute of Technology, Zhejiang University, Ningbo, 315100, China

    Sheng Hu, Weirui Zhao & Lehe Mei

  3. Hangzhou Huadong Medicine Group Co. Ltd, Hangzhou, 310011, China

    Jiaqi Mei

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  1. Yujiao Hua
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Correspondence to Jun Huang or Lehe Mei.

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Hua, Y., Lyu, C., Liu, C. et al. Improving the Thermostability of Glutamate Decarboxylase from Lactobacillus brevis by Consensus Mutagenesis. Appl Biochem Biotechnol 191, 1456–1469 (2020). https://doi.org/10.1007/s12010-020-03283-0

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